The long-range objective is to contribute to an understanding of electrogenic Na/Ca exchange which links [Nai] and, partially, membrane potential to [Cai] and is therefore an important determinant of myocardial cell function. Specific Aim 1 will test two hypotheses: 1) The monomer of the exchanger has a mobility on SDS-PAGE equivalent to 150 kDa; 2) The molecular weight of the protein complex responsible for Na/Ca exchange is about 353 kDa and is an oligomer composed of identical protein subunits. A highly enriched Na/Ca exchange preparation containing the 150 kDa protein and three proteolytic products will be used. Experimental approaches include attempts to separate the 150 kDa protein from the other three by chromatographic and electrophoretic means, to test the ability of the cDNA encoding for the 150 kDa protein to express Na/Ca exchange in cell lines, and crosslinking studies to determine the functional molecular weight of the exchanger. Subsequent studies will focus on deducing the primary structure of the protein from the corresponding structure of its cDNA. Specific Aim 2 will test three hypotheses 1) Membrane potential affects Na/Ca exchange at the transport step as opposed to the steps where Na and Ca bind; 2) Under some circumstances, Na and Ca movements are partially to totally uncoupled; 3) Na and Ca transport occurs through a sequential mechanism. Experimental approaches include measuring Na/Ca exchange versus Ca and Na at various membrane potentials, comparing specific activities of Na/Ca exchange and a potential-dependent Ca flux observed in the absence of Na but inhibited by Na, and assessing the ability of Ca binding sites to become inaccessible after exposure to Ca. Specific Aim 3 will test one hypothesis: The cation binding domain carries one excess negative charge when occupied by Ca and is neutral when occupied by three Na. Experimental approaches include testing predictions about secondary structure of the protein by antibodies raised against synthetic peptides corresponding to known segments of the protein, testing juxtaposition of putative extramembranal loops and transmembrane segments by crosslinking studies, attempts to define the opening of the groove or channel leading to the binding domain for Ca and Na with monoclonal antibodies, use of site- directed mutagenesis to test predictions of amino acid residues located in the binding domain, and 113-Cd NMR studies to probe the nature and dynamics of the binding domain.